Enhanced detection and serotyping of foot‐and‐mouth disease virus serotype O, A, and Asia1 using a novel multiplex real‐time RT‐PCR

Abstract Rapid and accurate detection and serotyping of foot‐and‐mouth disease (FMD) virus (FMDV) is essential for implementing control policies against emergent FMD outbreaks. Current serotyping assays, such as VP1 reverse transcription‐polymerase chain reaction (RT‐PCR)/sequencing (VP1 RT‐PCR/sequencing) and antigen detection enzyme‐linked immunosorbent assay (ELISA), have problems with increasing serotyping failure of FMDVs from FMD outbreaks. This study was conducted to develop a multiplex real‐time RT‐PCR for specific detection and differential serotyping of FMDV serotype O, A, and Asia 1 directly from field clinical samples. Primers and probes were designed based on 571 VP1 coding region sequences originated from seven pools. Multiplex real‐time RT‐PCR using these primers and probes demonstrated serotype‐specific detection with enhanced sensitivity compared to VP1 RT‐PCR/sequencing for reference FMDV (n = 24). Complete serotyping conformity between the developed multiplex real‐time RT‐PCR and previous VP1 RT‐PCR/sequencing was demonstrated using FMDV field viruses (n = 113) prepared in cell culture. For FMDV field clinical samples (n = 55), the serotyping rates of multiplex real‐time RT‐PCR and VP1 RT‐PCR/sequencing were 92.7% (51/55) and 72.7% (40/55), respectively. Moreover, the developed multiplex real‐time RT‐PCR demonstrated improved FMDV detection (up to 33.3%) and serotyping (up to 67.7%) capabilities for saliva samples when compared with 3D real‐time RT‐PCR and VP1 RT‐PCR/sequencing, during 10 days of challenge infection with FMDV serotype O, A, and Asia 1. Collectively, this study suggests that the newly developed multiplex real‐time RT‐PCR assay may be useful for the detection and differential serotyping of FMDV serotype O, A, and Asia 1 in the field.


INTRODUCTION
Foot-and-mouth disease (FMD) is a highly contagious transboundary disease of domestic and wild cloven-hoofed animals caused by the foot-and-mouth disease virus (FMDV). FMD outbreaks are associated with considerable economic losses due to their effect on trade and decreased animal productivity (World Organisation for Animal Health [OIE], 2019). FMDV belongs to the genus Aphthovirus of the family Picornaviridae and consists of seven genetically and immunologically distinct serotypes (O, A, Asia 1, C, Southern African Territories [SAT] 1, SAT 2, and SAT 3). These serotypes have multiple topotypes and genetic lineages (Knowles & Samuel, 2003).
Based on genetic and antigenic analyses, FMDVs throughout the world have been geographically divided into seven regional pools Because VP1 coding sequences vary greatly depending on the FMDV serotype, serotyping can be achieved directly by RT-PCR of the serotype-specific VP1 coding region without sequencing (Alexandersen et al., 2000;Callens & de Clerq 1997;Le et al., 2011;Vangrysperre & de Clercq, 1996) However, these serotype-specific RT-PCRs are troublesome and unsuitable for routine use because of their low sensitivity and specificity (Reid et al., 2001). Therefore, several realtime RT-PCR assays have been developed for single serotype-specific detection (Jamal & Belsham, 2015;Reid et al., 2014). Nevertheless, their serotyping methods were designed to specifically detect serotype O, A, and Asia 1 FMD viruses circulating in the Middle East (Reid et al., 2014) or West Eurasia (Jamal & Belsham, 2015), and not proved for detection of genetically variable FMDVs under the same serotypes circulating in other pools, which may lead to failure or delay in making serotype-specific preventive controls including emergent vaccination against unprecedented serotypes of FMD outbreaks.
Therefore, this study was conducted to develop a multiplex realtime RT-PCR for specific detection and differential serotyping of FMDV serotype O, A, and Asia 1 from field clinical samples during FMD outbreak with enhanced performance.

3D real-time RT-PCR
The 3D real-time RT-PCR assay for FMDV was performed with 3D gene-specific primers and probes according to previously described OIE-recommended methods (Callahan et al., 2002), which are currently in use for the molecular diagnosis of FMD at the Animal and Plant Quarantine Agency (APQA

VP1 RT-PCR/sequencing
RT-PCR for FMDV VP1 coding region sequencing was performed with a primer set (forward primer: 5'-AGYGCYGGYAARGAYTTTGA-3' , reverse primer: 5'-CATGTCYTCYTGCAT CTGGTT-3') designed to anneal within the VP3 coding region (forward primer) and the 2B coding region (reverse primer) to amplify the VP1 full-length coding region as shown in the previous study (Le et al, 2012). RT-PCR was performed using a commercial one-step RT-PCR kit (Qiagen, Hilden, Germany).

Design of primers and probes
A total of 571 full-length VP1 coding sequences classified into seven different serotypes of FMDVs of seven pools (pool 1, n = 410; pool 2, n = 40; pool 3, n = 60; pool 4, n = 27; pool 5, n = 8; pool 6, n = 10; pool 7, n = 16), were collected from GenBank of the National Center for Biotechnology Information or by sequencing clinical samples of FMD outbreaks in our laboratory. The VP1 coding region sequences were aligned using BioEdit (http://www.mbio.ncsu. edu/bioedit/bioedit) and analyzed collectively. Following the analyses, VP1 conserved regions were identified as targets for primers and probes design for each serotype. Several primer and probe sets were designed and evaluated in multiple possible combinations to detect the homologous FMDV serotype to select the best-performing primers and probes. Bioinformatic analysis was conducted using Fast-PCR software 6.0.04 (PrimerDigital Ltd., Finland) for all the primers and probes to verify their specificity. These in-silico evaluations were based on all available sequences of FMDV obtained from GenBank and our laboratory. Through the in-silico PCR results, each set of primers and probes (Table 1) for the multiplex real-time RT-PCR was proven to have high specificity for the current circulating FMDVs. All primers and probes were synthesized by Bionics.

Real-time RT-PCR protocol
The multiplex real-time RT-PCR assay for detection and serotyping of FMDV was performed using serotype-specific primers and probes according to the following procedure, using a CFX96 Touch real-time

Serotyping using field viruses prepared in cell culture
Of the 113 field viruses prepared from LFBK-α V β 6 cells, 92 and 21 were identified as FMDV serotype O and A, respectively, by the VP1 RT-PCR/sequencing (Table 4), which were in agreement with the serotypes determined by multiplex real-time RT-PCR.

Comparison of serotyping performance for clinical field samples
To evaluate serotyping performance, serotyping was conducted for clinical field samples (n = 55), and field viruses (n = 45) successfully isolated in cell culture, which was inoculated with the same clinical field samples, using VP1 RT-PCR/sequencing and multiplex  (Table 5). Among the serotypes determined by multiplex real-time RT-PCR and VP1 RT-PCR/sequencing in field clinical samples, the serotype consistency rate between the two assays was 90% (36/40) due to mixed serotypes determined only by the multiplex real-time RT-PCR. For the serotypes determined by multiplex real-time RT-PCR for field clinical samples and VP1 RT-PCR/sequencing for viral isolates (after viral isolation), the serotype consistency rate between two assays was 100% (45/45). Meanwhile, among the serotypes determined by VP1 RT-PCR/sequencing for field clinical samples and viral isolates, the serotype consistency rate was 94.7% (36/38), due to mixed serotypes for two viral isolates.

Detection and serotyping of saliva from FMDV infected pigs
Pigs were challenged with FMDV strains of serotype O, A, and Asia 1, and all of the FMDV-infected pigs showed typical clinical signs of FMD from 1-3 days-post challenge (dpc) and viremia (data not shown).
In the serotype O infection group, FMD viral RNAs were detected in the saliva of all pigs 1 to 10 dpc, using 3D real-time RT-PCR. Saliva was successfully determined as serotype O for all pigs at 3 and 4 dpc, but only two pigs at 6 and 7 dpc using VP1 RT-PCR/sequencing, while saliva of all pigs was determined as the serotype O using multiplex realtime RT-PCR throughout 1 to 10 dpc. In the serotype A infection group, the viral RNAs were detected in the saliva of one pig at 1, 7, and 8 dpc, two pigs at 2 and 9 dpc, and all pigs at 3 to 6 dpc, using 3D real-time RT-PCR. Saliva was successfully serotyped as serotype A for two pigs at 2 dpc and all pigs at 3 to 6 dpc using VP1 RT-PCR/sequencing, while saliva of only one pig at 7 and 8 dpc, two pigs at 1 dpc, and all pigs at 2 to 6 dpc and 9 to 10 dpc was serotyped as serotype A using multiplex real-time RT-PCR. In the serotype Asia 1 infection group, the viral RNAs were detected in one pig at 1 dpc, two pigs at 7 dpc, and all pigs at 2 to 6 dpc using 3D real-time RT-PCR. Saliva was serotyped as serotype Asia 1 for all pigs at 2 to 6 dpc using VP1 RT-PCR/sequencing, while saliva of two pigs at 8 and 10 dpc and all pigs at 1 to 7 dpc and 9 dpc was serotyped as serotype Asia 1 using multiplex real-time RT-PCR ( Figure 3). Callens & de Clercq 1997; Le et al., 2011;Vangrysperre & de Clercq, 1996) and real-time RT-PCR (Jamal & Belsham, 2015;Reid et al., 2014). Nevertheless, these assays are not widely used for serotyping FMDV.
As mentioned earlier, low sensitivity and complicated processes of the current standard assays for serotyping have caused some problems as follows. First, the low sensitivity may lead to failure of serotyping for samples containing low amount of field viruses. There have been reports regarding an increasing rate of untyped FMD suspected clinical samples from 9.2 to 15.2% between 2015 and 2020 (OIE/FAO

Reference Laboratory Network for Foot-and-Mouth Disease Annual
Reports 2015-2020), in which FMDV genomes were positive with RT-PCR. The failure of serotyping was mainly due to the low sensitivity of the RT-PCR, which was supported by the result that sequencing following VP1 RT-PCR had low serotyping sensitivity (10 2 to 10 4 TCID 50 /mL) as investigated in this study (Table 3) (Table 5) of two to three different serotypes, in comparison to VP1 RT-PCR/sequencing. In particular, as shown in and Asia 1) in comparison to the current assays. Nonetheless, the specificity of the novel multiplex real-time RT-PCR should be continuously evaluated since the assay targets the most variable region (VP1 coding region sequence) on FMDV genome, which is highly vulnerable to genetic variation by evolutionary pressure (Carrillo et al., 2005). Even more, it would be essential to further evaluate the assay for a practical field application using a large number of FMD clinical samples from endemic regions.

CONFLICT OF INTEREST
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

ETHICAL STATEMENTS
The animal experiments were supervised by the Institutional Animal Care and Use Committee of the APQA (IACUC number: 2018(IACUC number: -125, 2020(IACUC number: -393 and 2021 in the Republic of Korea and performed in accordance with the regulations and guidelines of this committee.

DATA AVAILABILITY STATEMENT
Primer and probe sequences are the primary data. A summary of standard curve and amplification curve data is also included; raw data are available upon request.